Method of assaying specimen substance by controlling dose of chemiluminescence

ABSTRACT

PCT No. PCT/JP96/00218 Sec. 371 Date Oct. 8, 1997 Sec. 102(e) Date Oct. 8, 1997 PCT Filed Feb. 2, 1996 PCT Pub. No. WO96/24044 PCT Pub. Date Aug. 8, 1996A method for assay of an analyte by use of a material labeled with a chemiluminescent substance, which comprises adding a quencher and/or decreasing the specific activity of a chemiluminescent substance labeled probe, thereby decreasing the quantity of chemiluminescence.

TECHNICAL FIELD

This invention relates to a method for assay of an analyte by use of amaterial labeled with a chemiluminescent substance. More specifically,the invention relates to a method for assay in a broader range bydecreasing the quantity of chemiluminescence.

BACKGROUND ART

Among methods for assay of an analyte in a test solution is one using aprobe labeled with a chemiluminescent substance. This method is widelyused as being capable of determining the amount of the analyte highlysensitively by measuring the quantity of chemiluminescence. This methodis useful for a suitable amount of the analyte. In the presence of alarge amount of the analyte (e.g. when many copies are produced by geneamplification such as polymerase chain reaction), however, the quantityof chemiluminescence by the method exceeds the determination limit of ameasuring device, making accurate assay impossible. Such samples withresults in excess of the upper assay limit have been determined againafter dilution of the test solution. This procedure is very laborious.Samples amplified by gene amplification, in particular, can causecontamination of the amplified product as a result of dilution. Utmostcare has been taken to avoid the contamination, further increasinglabor. To broaden the range of assay without diluting the sample, therehas been no choice but to wait for an improvement in the measuringdevice.

We, the inventors, have found that the foregoing problems with thedetermination of a sample beyond the assay limits can be solved bydecreasing the quantity of chemiluminescence, without requiring alaborious operation such as the dilution of the sample, or animprovement in the measuring device. This finding has led us toaccomplish this invention.

DISCLOSURE OF THE INVENTION

The present invention is a method for assay of an analyte by use of amaterial labeled with a chemiluminescent substance, which comprisesdecreasing the quantity of chemiluminescence. The invention furtherprovides a method for assay which comprises adding a quencher and/ordecreasing the specific activity of a chemiluminescent substance labeledprobe.

The assay of the analyte by use of a material labeled with achemiluminescent substance, indicated above, refers, for example, toreacting a sample containing an analyte with a material labeled with achemiluminescent substance, and measuring the quantity ofchemiluminescence of the conjugate to detect or determine the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of Phenol Red addition on the quantitativeamplification and detection of HBV template in the serum; and

FIG. 2 shows the effect of unlabeled probe addition on the quantitativeamplification and detection of HBV template in the serum.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Decreasing the Quantity of Chemiluminescence by Addition of aQuencher

The quencher used in the present invention may be any substance whichcan quench chemiluminescence. For example, it includes color matters andindia ink (drops of india ink, supernatant of india ink). Examples ofthe color matters are Crystal Violet, Bromophenol Blue, carminic acid,Chlorophenol Red, hematoxylin, Bromophenol Purple, Bromophenol Red,rosolic acid, Phenol Red, Cresol Red, and Methacresol Red.

When the color matter is used as the quencher, the concentration of thecolor matter at measurement of chemiluminescence may be in the range of0.01 to 10%, preferably 0.01 to 1%, although it differs depending on thecolor matter used. When india ink is used as the quencher, on the otherhand, its amount at measurement of chemiluminescence may be in the rangeof 0.01 to 50%, preferably 1 to 20%, based on the amount of the testsolution. The quencher may be added at any time before measurement ofchemiluminescence. For example, it may be added either before or afterthe reaction between the analyte and the labeled probe.

(2) Decreasing the Quantity of Chemiluminescence by Decreasing theSpecific Activity of a Chemiluminescent Substance Labeled Probe

To decrease the specific activity of a labeled probe in the presentinvention, an unlabeled probe is added to the labeled probe. Theunlabeled probe may be added in an amount in the range of 0.1 to 10⁵,preferably 10 to 10³, with respect to 1 of the labeled probe.

(3) Combining the Method (1) and the Method (2)

In the present invention, the addition of the quencher and the reductionof the specific activity of the chemiluminescent substance labeled probemay be combined. The conditions for use of both methods in thiscombination follow the above-described ranges.

The use of the assay method according to the present invention enablesthe quantity of chemiluminescence to be measured accurately, even whenthe analyte in the sample solution is present in so large an amount asto exceed the assay limit. For instance, the analyte can be easilydetected or determined quantitatively from a product produced by geneamplification of genetic information (DNA or RNA) on a microorganism orcell. This action can be confirmed by Examples 1 to 4 to be offeredlater on.

In the method involving the addition of the quencher, not only positivesignals, but also background (noise) levels are decreased. This decreasein the quantity of chemiluminescence permits the quantitativemeasurement of strongly positive signals, while the decrease in thebackground (noise) levels makes discrimination of weakly positivesignals possible. In short, the addition of the quencher enables theassay of a highly positive sample without affecting the determination ofa weakly positive sample. This action can be confirmed by Examples 1 to3 to be offered later on.

By using the present invention to decrease the quantity ofchemiluminescence, it becomes possible to detect or quantitativelydetermine a highly positive sample or a sample beyond the assay limits.The method of adding the quencher, in particular, reduces the background(noise) level as well. Thus, a highly positive sample can be measuredwithout influence on the assay of a weakly positive sample. Hence, themethod of the present invention proves to be an excellent method whichcan broaden the range of assay without diluting the sample or improvingthe measuring device.

EXAMPLES

The present invention will now be described in more detail withreference to Examples, whose descriptions do not limit the invention.

Example 1

Method

Five μl of human serum containing HBV-DNA sequence (Galibert, F.,Mandart, E., Fitioussi, F., Tiollais, P. and Charnay, P., Nature 281,646-650 (1979))(50 to 5,000 copies per amplification) was mixed with 20μof an alkaline solution (pH 13), followed by heating for 5 minutes at97° C. At room temperature, the mixture was allowed to cool for 10minutes, and then neutralized with a buffer. Two kinds of primers wereadded, and annealing was performed at room temperature. After DNA andRNA polymerases were added, gene amplification (using the methoddescribed in Officially Published Japanese Patent Gazette No. 500759/92)was carried out at 37° C. The amplification product and an acridiniumester labeled probe were hybridized at 60° C., whereafter theamplification product was detected by the HPA method (Arnold J R, L. J.,Hammond, P. W., Wiese, W. A. and Nelson, N. C., Clinical Chemistry 35,1588-1594 (1988)). In detecting the amplification product by themeasurement of chemiluminescence, the effect of addition of Phenol Redwas investigated. Phenol Red was added in an amount of 0.05% to a testsolution for chemiluminescence measurement to measure the quantity ofchemiluminescence. The results of measurement were compared with theresults obtained from the testing solution containing no Phenol Red. Theresults are shown in FIG. 1.

Discussion

As shown in FIG. 1, the Phenol Red-free sample nearly reached the assaylimit (saturation value) at about 500 GE (genome equivalents)/AMP, abovewhich the quantity of chemiluminescence became no more linear. With thePhenol Red-containing sample, the quantity of chemiluminescence remainedlinear even at 5,000 GE/AMP. This means that the addition of Phenol Redmade the assay of 500 GE/AMP or more possible. Compared with the PhenolRed-free sample, the Phenol Red-containing sample markedly reduced thebackground (noise) level (DNA content=0), thus permitting the assay of aweakly positive sample (about 50 GE/AMP). In other words, the additionof Phenol Red enabled the assay of a highly positive sample withoutaffecting the determination of a weakly positive sample.

Example 2

Method

In detecting the amplification product obtained by gene amplification asin Example 1, various amounts of Phenol Red were added to study theeffect of Phenol Red addition. Phenol Red was added in an amount of0.025 to 0.2% to a test solution for chemiluminescence measurement tomeasure the quantity of chemiluminescence. The results of measurementwere compared with the results obtained using the testing solutioncontaining no Phenol Red. The results are shown in Table 1.

                  TABLE 1    ______________________________________    Effect of Phenol Red at determination of quantity of    chemiluminescence               Positive    Phenol Red sample      Negative  Positive/    concentration               (50         sample    negative    (%)        GE/AMP)     (0 GE/AMP)                                     ratio    ______________________________________    0          237340      967       245    0.025      73120       239       306    0.05       41706       172       242    0.1        19332       114       170    0.2        7571        89        85    ______________________________________

Discussion

As shown in Table 1, the quantity of chemiluminescence from the positivesample and the quantity of chemiluminescence (background) from thenegative sample decreased in a manner dependent on the amount of PhenolRed added. These findings demonstrate that the inventive method isavailable in a wide range of phenol Red concentrations.

Example 3

Method

Tests were conducted in the same way as in Example 2, except thatcommercially available india ink was used instead of Phenol Red.

To a test solution for chemiluminescence measurement, 1.25 to 10% byvolume of india ink was added, and the quantity of chemiluminescence wasmeasured. The results are shown in Table 2 in comparison with theresults of measurement of the test solution free from india ink.

                  TABLE 2    ______________________________________    Effect of india ink at determination of    quantity of chemiluminescence    Amount of  Positive    india ink  sample      Negative  Positive/    added      (50         sample    negative    (% by volume)               GE/AMP)     (0 GE/AMP)                                     ratio    ______________________________________    0          291638      1689      173    1.25       30902       218       142    2.5        21292       119       179    5          6609        63        105    10         5172        43        120    ______________________________________

Discussion

As shown in Table 2, the quantity of chemiluminescence from the positivesample and the quantity of chemiluminescence (background) from thenegative sample decreased in a manner dependent on the amount of indiaink added. These findings demonstrate that the inventive method can beused in a wide range of india ink concentrations.

Example 4

Method

Gene amplification was performed in the same manner as in Example 1.When the amplification product and an acridinium ester labeled probewere hybridized at 60° C., an unlabeled probe in various amounts wasadded. The amounts of the unlabeled probe were 10 to 1,000 relative to 1of the labeled probe. After hybridization, the quantities ofchemiluminescence in the samples were measured by the HPA method. Theresults are shown in FIG. 2 in comparison with the results ofmeasurement of the test solution free from the unlabeled probe.

Discussion

As shown in FIG. 2, the unlabeled probe-free sample nearly reached theassay limit (saturation value) at about 500 to 5,000 GE/AMP, above whichthe quantity of chemiluminescence became no more linear. The unlabeledprobe-containing samples, on the other hand, showed decreases in thequantity of chemiluminescence in a manner dependent on the amount of theunlabeled probe added. When the amount of the unlabeled probe addedrelative to the labeled probe was 100(unlabeled probe):1(labeled probe)or more, assay was possible even at about 500,000 GE/AMP. Thus, theaddition of the unlabeled probe permitted assay of 500 to 500,000 GE/AMPor more.

We claim:
 1. A method for assay of an analyte which comprises the steps of:(1) providing a first probe that specifically binds to said analyte wherein said first probe is labeled with a chemiluminescent substance; (2) adding an unlabeled, second probe that specifically binds to said analyte; (3) contacting said first labeled probe and said unlabeled second probe with said analyte; (4) quantitatively measuring a chemiluminescent signal, wherein said unlabeled second probe decreases the chemiluminescent signal by decreasing a specific activity of the chemiluminescent substance of said first labeled probe; (5) adding a quencher to said analyte before said measuring step to decrease said chemiluminescent signal and decrease background noise levels of said chemiluminescent signal; and (6) correlating said chemiluminescent signal to the presence of said analyte.
 2. The method of claim 1, wherein said quencher is at least one member selected from the group consisting of color matter and india ink.
 3. The method of claim 1, wherein said unlabeled probe is added in an amount of from 10 to 10³ parts per one part of the labeled probe.
 4. The method of claim 2, wherein said color matter is selected from the group consisting of Crystal Violet, Bromophenol Blue, carminic acid, Chlorophenol Red, hematoxylin, Bromophenol Purple, Bromophenol Red, rosolic acid, Phenol Red, Cresol Red, and Methacresol Red. 